JP7046250B2 - Combination of TGFβ and CDK inhibitors for cancer treatment - Google Patents

Description

The present invention relates to combination therapies useful in the treatment of cancer. In particular, the present invention relates to a method for treating cancer by administering a TGFβ inhibitor in combination with a CDK inhibitor. Pharmaceutical uses of the combinations of the invention are also described.

TGFβ signaling is an emerging pathway in cancer progression and has a role in the regulation of immune responses and in many other cancer pathways, including metastasis and angiogenesis. Increased expression of TGFβ by tumor cells and stromal cells in the tumor microenvironment, as well as activation of intracellular signaling of TGFβ receptors, has been observed in many cancers (Massage J. TGFbeta in Cancer. Cell 2008). , 134 (2): 215-30; Neuzillet C, Tijeras-Laballand A, Cohen R et al., Targeting the TGFβ passway for cancer cell therapy.Pharmacol Ther 2015, 147: 22-31). The TGFβ signaling pathway can be activated upon interaction of a dimeric TGFβ ligand with its specific cell surface transmembrane serine / threonine kinase receptor. Activated TGFβ ligands interact with the TGFβ type II receptor (TGFβR2), which directs the TGFβ type I receptor (TGFβR1, also known as activin receptor-like kinase (ALK5)) to specific serine and threonine residues. (Principe DR, Doll JA, Bauer J et al., TGF-β: duality of faction betareceptor presentation and carcinogenesis.J NatlCanesis. J NatlCancer Activated TGFβR1 then phosphorylates SMAD2 and SMAD3, which can then aggregate into a complex with SMAD4, translocate to the nucleus, where in the nucleus this complex regulates the expression of the TGFβ target gene. (Massage J. TGFbeta in Cancer. Cell 2008, 134 (2): 215-30). In addition to SMAD signaling, non-SMAD signaling can also be initiated downstream of the TGFβ receptor, thereby phosphoinocitide 3-kinase (PI3K), c-Jun N-terminal kinase (JNK), and extracellular. Activation of various pathways such as signal-regulated kinase (P38 / ERK), mitogen-activated protein (MAP) kinase can be brought about (Mu Y, Gudy SK, Landstrom M. Non-Smad signing paces. Cell Tissue Res. 2012, 347 (1): 11-20).

Activation of the TGFβ pathway in cancer cells leads to epithelial-mesenchymal transition (EMT), in which epithelial cells lose their apical polarity and intercellular adhesion to become highly mobile mesenchymal cells, thereby resulting in metastasis. Can trigger. In addition to its importance in tumor cell migration and metastasis, EMT has also been associated with the avoidance of immune surveillance mechanisms by tumor cells (Akaly I, Janji B, Hasmim M et al., Epithelial-to-mesenchymal transition and autophology in breast carcinoma tumor escape from T-cell-mediated lysys. Cancer Res 2013, 73 (8): 2418-27). TGFβ is a potent immunosuppressant against both dendritic cells, macrophages, natural killer cells, and both natural and adaptive immune cells, including CD4 + and CD8 + T cells. Conversely, TGFβ has a key role in stimulating the differentiation of immunosuppressive regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSCs) (Akalay I, Janji B, Hasmim M et al., Epithelial). -To-mesenchymal transition and autophagy induction in breath carcinoma mapromote escape from T-cell-mediated lysys. Cancer Res 2013, 738 (27, 1827).

The TGFβ pathway has a key role in disease progression and resistance to treatment in a wide range of tumors (Neuzillet C, Tijeras-Raballand A, Cohen R et al., Targeting the TGFβ passway for cancer20 for cancer. 147: 22-31; Colak S, Ten Disease P. Targeting TGF-β signing in cancer. Trends in Cancer 2017, 3 (1): 56-71). High TGFβ signature and EMT gene expression are found in a variety of tumors (Mak MP, Tong P, Diao L et al., A Patient-Derived, Pan-Cancer EMT Signature Epithelial-Molecular Alterment Transition.Clin Cancer Res 2016; 22 (3): 609-20).

TGFβ localizes T cells around the tumor or in the stroma by inducing the expression of extracellular matrix (ECM) proteins and by suppressing the expression of chemokines and cytokines required for tumor infiltration of T cells. ECM-dense reactive stroma and infiltrative phenotype-free T cells are important regulators of the tumor microenvironment (Heged PS, Caranikas V, Evers S. The Where, the). When, and the How of Infiltration For Cancer Immunotherapies in the Era of Checkpoint Infiltration.Clin Cancer Res 2016, 22 (8): 1865-74).

Compound, 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropane-2-yl) nicotinamide ("PF-60952229") Alternatively, also referred to as "PF-2229"), is a potent and selective TGFβ (transforming growth factor beta) inhibitor having the following structure:

PF-06952229 and pharmaceutically acceptable salts thereof are disclosed in WO2015 / 103355 and US Pat. No. 10,030,004. The contents of each of these aforementioned references are incorporated herein by reference in their entirety.

Cyclin-dependent kinases (CDKs) are important cellular enzymes that play an essential role in the regulation of eukaryotic cell division and proliferation. The catalytic unit of a cyclin-dependent kinase is activated by a regulatory subunit known as a cyclin. At least 16 mammalian cyclins have been identified (Johnson DG, Walker CL. Cyclins and Cell Cycle Checkpoints. Annu. Rev. Pharmacol. Toxicol. (1999) 39: 295-312). Cyclin B / CDK1, cyclin A / CDK2, cyclin E / CDK2, cyclin D / CDK4, cyclin D / CDK6, and similar other heterodyne are important regulators of cell cycle progression. Further functions of cyclin / CDK heterodyne include the regulation of transcription, DNA repair, differentiation, and apoptosis (Morgan DO. Cyclin-dependent kinases: engineers, cells, and microprocessors. Annu. Rev. Cell. Dev. (1997) 13: 261 to 291).

Cyclin-dependent kinase inhibitors have been demonstrated to be useful in the treatment of cancer. Increased or temporarily aberrant activation of cyclin-dependent kinases has been shown to result in the development of human tumors, which are generally either the CDK protein itself or its regulators. Related to alterations (Cordon-Cardo C. Mutations of cell cycle regulators: biological and cellular impedances for human neoplasmia. Am.J. Pathol. (1995) 147: J. Pathol. (1995) 147: cancer: new targets for protein.Nat.Med. (1995) 1: 309-320; Hall M, Peters G. Genetic alterations of cyclins, cyclin-dependent kinase. ) 68: 67-108). Amplification of regulatory subunits of CDKs and cyclins, as well as mutations, gene deletions, or transcriptional silencing of endogenous CDK inhibitors have also been reported (Smalley et al., Identity of a novel subgroup of melanoma with KIT / cyclin). -Genedent kinase-4 overexpression.Cancer Res (2008) 68: 5743-52).

Clinical trials for the CDK4 / 6 inhibitors palbociclib, ribociclib, and abemaciclib have been conducted for breast and other cancers, either alone or in combination with other therapeutic agents. Palbociclib, ribociclib, and abemaciclib are hormone-accepted in certain patients in combination with an aromatase inhibitor such as letrozole in the first-line setting and in combination with fulvestrant in the second-choice or subsequent treatment. Approved for the treatment of body (HR) -positive, human epithelial growth factor receptor 2 (HER2) -negative, advanced or metastatic breast cancer (O'Leary et al., Treating cancer with selective CDK4 / 6 inhbitors. Nature Reviews (2016) 13: 417-430). CDK4 / 6 inhibitors have been shown to have significant clinical efficacy in ER-positive metastatic breast cancer, but like other kinases, their effects over time by the development of primary or acquired resistance. Can be limited.

Overexpression of CDK2 is associated with abnormal regulation of the cell cycle. The cyclin E / CDK2 complex has important roles in the regulation of G1 / S translocation, histone biosynthesis, and centrosome replication. Progressive phosphorylation of Rb by cyclin D / Cdk4 / 6 and cyclin E / Cdk2 releases the G1 transcription factor E2F and promotes the initiation of S phase. Activation of cyclin A / CDK2 during early S phase promotes phosphorylation of endogenous substrates that allow DNA replication and E2F inactivation for completion of S phase (Asghar et al., The history and future). of future of targeting cyclin-dependent kinases in cancer therapy, Nat. Rev. Drag. Discov. 2015, 14 (2): 130-146). Cyclin E, a CDK2 regulatory cyclin, is frequently overexpressed in cancer. Amplification or overexpression of cyclin E has long been associated with poor outcomes of breast cancer (Keyomarsi et al., Cyclin E and surveillance in patients with breath cancer. N Engl J Med. (2002) 347: 1566. Overexpression of cyclin E2 (CCNE2) has been associated with endocrine therapy resistance in breast cancer cells, and inhibition of CDK2 has been reported to restore susceptibility to tamoxiphen or CDK4 inhibitors in tamoxiphen-resistant cells and CCNE2-overexpressing cells. and that (Caldon, et al., Cyclin E2 overexpression is associated with endocrine resistance but not insensitivity to CDK2 inhibition in human breast cancer cells.Mol.Cancer Ther (2012) 11:. 1488 ~ 99; Herrera-Abreu et al., Early Adaptation and Acquired Resistance to CDK4 / 6 inhibition in Estrogen Receptor-Positive Breast Cancer, Cancer Res. (2016) 76: 2301-2313). Amplification of cyclin E has also been reported to contribute to trastuzumab resistance in HER2 + breast cancer (Scultriti et al., Cyclin E amplification / ovation expression is a mechanism of trastuzumab resistance : 3761-6). Cyclin E overexpression has also been reported to have a role in basal cell-like breast cancer and triple-negative breast cancer (TNBC), as well as in inflammatory breast cancer (Elsawaf & Sinn, Triple Negative Breast Cancer: Clinical and Historical Breast Cancer). Care (2011) 6: 273-278; Alexander et al., Cyclin E overexpression as a biomarker for combination treatment treatment strategies in inflammatory breastcancer: 197tcan

Palbociclib, ie 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazine-1-yl-pyridin-2-ylamino) -8H-pyrido [2,3-d] pyrimidine-7-one (PD) -0332991) is a potent and selective inhibitor of CDK4 and CDK6 having the following structure:

Palbociclib is described in WHO Drag Information, Vol. 27, No. 2, p. 172 (2013). Parvocyclib and its pharmaceutically acceptable salts are described in International Publication No. WO2003 / 062236 and US Pat. No. 6,936,612, US Pat. No. 7,208,489, and US Pat. No. 7,456,168; WO 2005/005426 and US Pat. No. 7,345,171 and US Pat. No. 7,863,278; WO 2008/032157 and US Pat. No. 7,781,583; and International Publication No. 7. It is disclosed in WO2014 / 128588. The contents of each of these aforementioned references are incorporated herein by reference in their entirety.

PF-06673600, ie 6- (difluoromethyl) -8-((1R, 2R) -2-hydroxy-2-methylcyclopentyl) -2- (1- (methylsulfonyl) piperidine-4-ylamino) pyrid. 3-d] Pyrimidine-7 (8H) -one is a potent and selective inhibitor of CDK2, CDK4, and CDK6 having the following structures:

PF-06783600 is disclosed in International Publication No. WO2018 / 033815, published February 22, 2018. The contents of this reference are incorporated herein by reference in their entirety.

Palbociclib, a selective CDK4 / 6 inhibitor, has been shown to be clinically effective in breast cancer (DeMichelle A, Clark AS, Tan KS et al., CDK 4/6 inhibitor palbociclib (PD-0332991) in Rb + advanced breast cancer: phase II activity, safety, and predictive biomarker assessment.Clin Cancer Res 2015,21 (5): 995 ~ 1001; Finn RS, Martin M, Rugo HS et al, Palbociclib and Letrozole in Advanced Breast Cancer.New Engl J Med 2016,375 (20): 1925 ~ 36; Cristofanilli M, Turner NC, Bondarenko I, et al., Fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone-receptor-positive, HER2-negative metastatic breast cancer that progressed on prevulious endocrine therapy (PALOMA-3): final analysis of the multicentre, double-blind, phase 3 random controlled trial. Ranchet Oncol 2016, 17 (4): 425-39), followed by first clinical efficacy Possible (Knudsen Eric S., Breast cancer Agnieszka K., The Strage Case of CDK4 / 6 Inhibitors: Mechanisms, Resistance, and Combination In preclinical studies, treatment of tumor cells with palbociclib induces expression of TGFβ and EMT gene signatures and enhances tumor cell invasiveness.

International Publication No. WO2015 / 103355 U.S. Pat. No. 10,030,004 International Publication No. WO2003 / 062236 U.S. Pat. No. 6,936,612 U.S. Pat. No. 7,208,489 U.S. Pat. No. 7,456,168 International Publication No. WO2005 / 005426 U.S. Pat. No. 7,345,171 U.S. Pat. No. 7,863,278 International Publication No. WO2008 / 032157 U.S. Pat. No. 7,781,583 International Publication No. WO2014 / 128588 International Publication No. WO2018 / 033815

Massage J. TGFbeta in Cancer. Cell 2008, 134 (2): 215-30 Neuzillet C, Tijeras-Laballand A, Cohen R et al., Targeting the TGFβ passway for cancer therapy. Pharmacol The 2015, 147: 22-31 Principe DR, Doll JA, Bauer J et al., TGF-β: duality of function benefiten tumor presentation and carcinogenesis. J Natl Cancer Inst 2014, 106 (2): djt369 Mu Y, Goody SK, Landstrom M. et al. Non-Smad signaling passingways. Cell Tissue Res 2012, 347 (1): 11-20 Akary I, Janji B, Hasmim M et al., Epithelial-to-mesenchymal transition and autophagy induction in breast cancer romote master-sequel. Cancer Res 2013, 73 (8): 2418-27 Colak S, Ten Dijke P.M. Targeting TGF-β signing in cancer. Trends in Cancer 2017, 3 (1): 56-71 Mak MP, Tong P, Diao L et al., A Patient-Derived, Pan-Cancer EMT Signature Ideas Global Molecule Alterations and Immune Target Element Clin Cancer Res 2016; 22 (3): 609-20 Hegde PS, Karanikas V, Evers S.A. The Who, the Who, and the How of Immunotherapy for Cancer Immunotherapy in the Era of Checkpoint Inspection. Clin Cancer Res 2016, 22 (8): 1865-74 Johnson DG, Walker CL. Cyclins and Cell Cycle Checkpoints. Annu. Rev. Pharmacol. Toxicol. (1999) 39: 295-312 Morgan DO. Cyclin-dependent kinases: engineers, blocks, and microprocessors. Annu. Rev. Cell. Dev. Biol. (1997) 13: 261-291 Cordon-Cardo C.I. Mutations of cell cycle regulations: biological and cellular implications for human neoplasmia. Am. J. Pathol. (1995) 147: 545-560 Karp JE, Border S. Molecule foundations of cancer: new targets for intervention. Nat. Med. (1995) 1: 309-320 Hall M, Peters G.M. Genetic alterations of cyclins, cyclin-dependent kinases, andCdk inhibitors in human cancer. Adv. Cancer Res. (1996) 68: 67-108 Smalley et al., Identity of a novel subgroup of melanoma with KIT / cyclin-dependent kinase-4 overexpression. Cancer Res (2008) 68: 5743-52 O'Learly et al., Treating cancer with selective CDK4 / 6 inhbitors. Nature Reviews (2016) 13: 417-430 Asghar et al., The history and future of future of targeting cyclin-dependent kinases in cancer therapy, Nat. Rev. Drug. Discov. 2015, 14 (2): 130-146 Keiyomarsi et al., Cyclin E and survival in patients with breast cancer. N Engl J Med. (2002) 347: 1566-75 Caldon et al., Cyclin E2 overexpression is assisted with endocrine response but not incentitivity to CDK2 inhibition inhuman cancer. Mol. Cancer The. (2012) 11: 1488-99 Herrera-Abreu et al., Early Adaptation and Accuired Response to CDK4 / 6 inhibition in Estrogen Receptor-Positive Breast Cancer, Cancer Res. (2016) 76: 2301-2313 Scultriti et al., Cyclin E amplifier / overexpression is a mechanism of trastuzumab resistance in HER2 + breath cancer patients, Proc Natl Acc. (2011) 108: 3761-6 Elsawaf & Sinn, Triple Negative Breast Cancer: Histological and Histological Correlations, Breast Care (2011) 6: 273-278 Alexander et al., Cyclin E overexpression as a biomarker for combination strategy strategies in inflammatory breast cancer, Oncotarget (2017) 8: 14897- WHO Drag Information, Vol. 27, No. 2, p. 172 (2013) DeMichele A, Clark AS, Tan KS et al., CDK 4/6 inhibitor palladium (PD-0332991) in Rb + advanced breast cancer: phase II inhibitory, saffy Clin Cancer Res 2015, 21 (5): 995-1001 Finn RS, Martin M, Rugo HS et al., Palbociclib and Letrozole in Advanced Breast Cancer. New Engl J Med 2016, 375 (20): 1925-36 Cristofanilli M, Turner NC, Bondarenko I, et al., Fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone-receptor-positive, HER2-negative metastatic breast cancer that progressed on prevulious endocrine therapy (PALOMA-3): final analysis of the multicentre, double-blind, phase 3 hormone controlled controlled trial. Lancet Oncol 2016, 17 (4): 425-39 Knudsen Eric S. , Wikkiewicz Agnieszka K.K. , The Strategy Case of CDK4 / 6 Inhibitors: Mechanisms, Strategy, and Combination Strategies. Trends Cancer 2017, 3 (1): 39-55 Stahl and Wermus, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002) Remington's Pharmaceutical Sciences, Mack Publishing Company, Easter, Pa. , 15th Edition (1975)

Improved combination therapies for the treatment of breast cancer, including breast cancer that is resistant to CDK inhibitors, have significant unresolved medical demands, and the identification of new combination regimens improves treatment outcomes. It is necessary to do.

Each of the embodiments described below can be combined with any other embodiment described herein that is consistent with it. Further, each of the embodiments described herein envisions, within its scope, a pharmaceutically acceptable salt of the compounds described herein. Accordingly, the expression "or pharmaceutically acceptable salt thereof" is implied in the description of all compounds described herein.

The embodiments described herein include administering to a patient in need thereof an amount of a TGFβ inhibitor and an amount of a CDK inhibitor for breast cancer, particularly HR positive, HER2-negative. , A method for treating advanced or metastatic breast cancer, wherein the combined amount is effective in treating the cancer. Further embodiments of this embodiment include administration of a third component, which is an aromatase inhibitor or fulvestrant.

A further embodiment described herein comprises administering to a patient in need thereof a synergistic amount of a TGFβ inhibitor in combination with a CDK inhibitor, breast cancer, particularly HR positive, HER2. Concerning methods for treating negative, advanced, or metastatic breast cancer. Further embodiments of this embodiment include administration of a third component, which is an aromatase inhibitor or fulvestrant.

Further embodiments described herein relate to a combination of TGFβ and CDK inhibitors for use in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. .. Further embodiments of this embodiment include administration of a third component, which is an aromatase inhibitor or fulvestrant.

Some embodiments described herein are TGFβ inhibitors and CDK inhibitors in the manufacture of agents for the treatment of breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding the use of agents. Further embodiments of this embodiment include the use of a third component, which is an aromatase inhibitor or fulvestrant.

Further embodiments described herein are synergistic of TGFβ and CDK inhibitors for use in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding various combinations. Further embodiments of this embodiment include combinations that also include a third component that is an aromatase inhibitor or fulvestrant.

Some embodiments described herein are synergistic amounts of TGFβ in the manufacture of agents for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. With respect to the use of inhibitors and CDK inhibitors. Further embodiments of this embodiment include the use of a third component, which is an aromatase inhibitor or fulvestrant.

In certain embodiments of the methods or uses of the invention, the TGFβ inhibitors are Garnicertibu, LY2109761, SB525334, SP505124, GW788388, LY364497, RepSox, SD-208, Bactoseltib, LY3280882, and 4- (2- (5- (5-). Chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropane-2-yl) nicotinamide (PF-609522229), or a pharmaceutically acceptable salt thereof. Selected from the group.

In certain embodiments of the methods or uses of the invention, the TGFβ inhibitor is 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1, 3-Dihydroxypropan-2-yl) nicotinamide (PF-60952229), or a pharmaceutically acceptable salt thereof.

In certain embodiments of the methods or uses of the invention, the CDK inhibitor is a CDK4 / 6 inhibitor or a CDK2 / 4/6 inhibitor.

In certain embodiments of the methods or uses of the invention, the CDK inhibitor is a CDK4 / 6 inhibitor.

In some embodiments of the methods or uses of the invention, the CDK4 / 6 inhibitor is selected from the group consisting of abemaciclib, ribociclib, and palbociclib, or pharmaceutically acceptable salts thereof.

In some embodiments of the methods or uses of the invention, the CDK4 / 6 inhibitor is palbociclib or a pharmaceutically acceptable salt thereof.

In certain embodiments of the methods or uses of the invention, the CDK inhibitor is a CDK2 / 4/6 inhibitor.

In some embodiments of the methods or uses of the invention, the CDK2 / 4/6 inhibitor is 6- (difluoromethyl) -8-((1R, 2R) -2-hydroxy-2-methylcyclopentyl) -2. -(1- (Methylsulfonyl) piperidine-4-ylamino) pyrido [2,3-d] pyrimidin-7 (8H) -one ("PF-06673600") or a pharmaceutically acceptable salt thereof.

The embodiments described herein are for a patient in need thereof in an amount of 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N-. Breast cancer, comprising administering (1,3-dihydroxypropan-2-yl) nicotinamide (PF-60952229) or a pharmaceutically acceptable salt thereof, and an amount of palbociclib or a pharmaceutically acceptable salt thereof. In particular, it relates to a method for treating HR-positive, HER2-negative, advanced or metastatic breast cancer, wherein the combined amounts are effective in treating breast cancer.

A further embodiment described herein is a synergistic amount of 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) for a patient in need thereof. Containing administration of -N- (1,3-dihydroxypropan-2-yl) nicotinamide (PF-609522229) or a pharmaceutically acceptable salt thereof in combination with palbociclib or a pharmaceutically acceptable salt thereof. It relates to a method for treating breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer.

Further embodiments described herein are 4- (2- (5-chloro-) for use in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. 2-Fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropan-2-yl) nicotinamide (PF-609522229) or a pharmaceutically acceptable salt thereof and palbociclib or its thereof. Concerning combinations with pharmaceutically acceptable salts.

Some embodiments described herein are 4- (2-(2) in the manufacture of agents for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. 5-Chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropan-2-yl) nicotinamide (PF-609522229) or a pharmaceutically acceptable salt thereof, And with respect to the use of palbociclib or its pharmaceutically acceptable salts.

Further embodiments described herein are 4- (2- (5-chloro-) for use in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. 2-Fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropan-2-yl) nicotinamide (PF-609522229) or a pharmaceutically acceptable salt thereof and palbociclib or its thereof. Concerning synergistic combinations with pharmaceutically acceptable salts.

Some embodiments described herein are synergistic amounts of 4 in the manufacture of agents for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. -(2- (5-Chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropan-2-yl) nicotinamide (PF-60952229) or its pharmaceuticals With respect to acceptable salts, and the use of palbociclib or its pharmaceutically acceptable salts.

Further embodiments described herein are combinations of TGFβ and CDK inhibitors for use in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. With respect to the combination, the CDK inhibitor is administered according to a non-standard clinical dosing regimen.

Further embodiments described herein are of TGFβ and CDK inhibitors in the manufacture of agents for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. For use, the CDK inhibitor is administered according to a non-standard clinical dosing regimen.

In embodiments of the methods or uses of the invention, the non-standard clinical dosing regimen is a non-standard clinical dose.

In embodiments of the methods or uses of the invention, the non-standard clinical dose is a low dose CDK inhibitor.

In embodiments of the methods or uses of the invention, the non-standard clinical dosing regimen is a non-standard dosing schedule.

In embodiments of the methods or uses of the invention, the non-standard dosing schedule is a continuous dosing schedule of CDK inhibitors.

In embodiments of the methods or uses of the invention, the CDK inhibitor is a CDK4 / 6 inhibitor.

In embodiments of the methods or uses of the invention, the TGFβ inhibitor is 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxy). Propane-2-yl) nicotinamide (PF-60952229) or a pharmaceutically acceptable salt thereof, and the CDK inhibitor is palbociclib or a pharmaceutically acceptable salt thereof.

The embodiments described herein are for a patient in need thereof in an amount of 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N-. Breast cancer, comprising administering (1,3-dihydroxypropan-2-yl) nicotine amide (PF-60952229) or a pharmaceutically acceptable salt thereof, and an amount of palbociclib or a pharmaceutically acceptable salt thereof. In particular, a method for treating HR-positive, HER2-negative, advanced or metastatic breast cancer in which palbociclib or a pharmaceutically acceptable salt thereof is administered according to a non-standard clinical dosing regimen and further. The combined amount relates to a method that is effective in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer.

Further embodiments described herein are 4- (2- (5-chloro-) for use in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. 2-Fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropan-2-yl) nicotinamide ("PF-609522229") or a pharmaceutically acceptable salt thereof and palbociclib. Or in combination with a pharmaceutically acceptable salt thereof, wherein palbociclib or a pharmaceutically acceptable salt thereof is administered according to a non-standard clinical dosing regimen.

Further embodiments described herein are 4- (2- (5- (5- (5-)) in the manufacture of agents for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. Chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropane-2-yl) nicotinamide (PF-609522229) or a pharmaceutically acceptable salt thereof, and palbociclib Or the use of a pharmaceutically acceptable salt thereof, wherein palbociclib or the pharmaceutically acceptable salt thereof is administered according to a non-standard clinical dosing regimen.

In embodiments of the methods or uses of the invention, the non-standard clinical dosing regimen is a non-standard clinical dose.

In embodiments of the methods or uses of the invention, the non-standard clinical dose is a low dose of palbociclib or a pharmaceutically acceptable salt thereof.

In embodiments of the methods or uses of the invention, the low dose of palbociclib or a pharmaceutically acceptable salt thereof is about 50 mg, about 75 mg, or about 100 mg once daily.

In embodiments of the methods or uses of the invention, the low dose of palbociclib or a pharmaceutically acceptable salt thereof is about 75 mg once daily.

In embodiments of the methods or uses of the invention, the low dose of palbociclib or a pharmaceutically acceptable salt thereof is about 100 mg once daily.

In embodiments of the methods or uses of the invention, the non-standard clinical dosing regimen is a non-standard dosing schedule.

In embodiments of the methods or uses of the invention, the non-standard dosing schedule is a continuous dosing schedule of palbociclib or a pharmaceutically acceptable salt thereof.

In embodiments of the methods or uses of the invention, the continuous dosing schedule for palbociclib or a pharmaceutically acceptable salt thereof is a full cycle of 21 days.

In embodiments of the methods or uses of the invention, the continuous dosing schedule for palbociclib or a pharmaceutically acceptable salt thereof is a full 28-day cycle.

In embodiments of the methods or uses of the invention, the non-standard dosing schedule is to administer palbociclib or a pharmaceutically acceptable salt thereof once daily for 14 consecutive days, followed by no treatment for 7 days. Including doing.

In embodiments of the methods or uses of the invention, the non-standard clinical dosing regimen is to administer approximately 75 mg of palbociclib or a pharmaceutically acceptable salt thereof once daily for 14 consecutive days, followed by 7 Includes leaving the day untreated.

The embodiments described herein are for a patient in need thereof in an amount of 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N-. Breast cancer, comprising administering (1,3-dihydroxypropan-2-yl) nicotine amide (PF-60952229) or a pharmaceutically acceptable salt thereof, and an amount of palbociclib or a pharmaceutically acceptable salt thereof. In particular, a method for treating HR-positive, HER2-negative, advanced or metastatic breast cancer in which palbociclib or a pharmaceutically acceptable salt thereof is administered according to a non-standard clinical dosing regimen and further. The combined amount relates to a method that is effective in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer.

Further embodiments described herein are 4- (2- (5-chloro-) for use in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. 2-Fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropan-2-yl) nicotinamide (PF-609522229) or a pharmaceutically acceptable salt thereof and palbociclib or its thereof. Concerning a combination of a pharmaceutically acceptable salt in which palbociclib or a pharmaceutically acceptable salt thereof is administered according to a non-standard clinical dosing regimen.

Further embodiments described herein are 4- (2- (5- (5- (5-)) in the manufacture of agents for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. Chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropane-2-yl) nicotinamide (PF-609522229) or a pharmaceutically acceptable salt thereof, and palbociclib Or the use of a pharmaceutically acceptable salt thereof, wherein palbociclib or the pharmaceutically acceptable salt thereof is administered according to a non-standard clinical dosing regimen.

The embodiments described herein are:
It relates to a synergistic combination of (a) a TGFβ inhibitor and (b) a CDK inhibitor.

Further embodiments described herein are:
It relates to a synergistic combination of (a) a TGFβ inhibitor and (b) a CDK inhibitor, wherein the constituents (a) and (b) are synergistic.

Further embodiments relate to pharmaceutical compositions of TGFβ inhibitors and CDK inhibitors for use in the treatment of breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer.

In embodiments of the combinations of the invention, TGFβ is 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropane-2-). Il) Nicotinamide (PF-60952229) or a pharmaceutically acceptable salt thereof.

In embodiments of the combination of the invention, the CDK inhibitor is a CDK4 / 6 inhibitor.

In embodiments of the combinations of the invention, the CDK4 / 6 inhibitor is selected from the group consisting of abemaciclib, ribociclib, and palbociclib, or pharmaceutically acceptable salts thereof.

In embodiments of the combinations of the invention, the CDK4 / 6 inhibitor is palbociclib or a pharmaceutically acceptable salt thereof.

In embodiments of the combination of the invention, the TGFβ inhibitor is 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropane-). 2-Il) Nicotinamide (PF-60952229) or a pharmaceutically acceptable salt thereof, and the CDK inhibitor is palbociclib or a pharmaceutically acceptable salt thereof.

FIG. 6 shows a survival curve of mice with CT26 tumors treated with the medium, PF-0332991, PF-06783600, PF-60952229, PF-06952229 and PD-0332991, or the combination of PF-60952229 and PF-06673600. be. Tumor volume in CT26 syngeneic tumor model 17 days post-treatment for medium, PF-06952229, PF-0332991, PF-06783600, PF-06952229 and PD-0332991 combination, or PF-069522229 and PF-067883600 combination. It is a figure which shows. These combinations have been shown to increase the inhibition of tumor growth. It is a figure which shows the tumor volume in the MCF-7 ER ⁺ breast cancer tumor model 21 days after the treatment about the medium, PF-06952229, PF-0332991, and the combination of PF-06952229 and PD-0332991. These combinations have been shown to increase the inhibition of tumor growth. Tumor volume in MCF-7 ER ⁺ breast cancer tumor model 21 days post-treatment for medium, PF-06952229, PF-0332991 and fulvestrant combination, and PF-06952229 and PD-0332991 and fulvestrant combination. It is a figure which shows. These combinations have been shown to increase the inhibition of tumor growth. The addition of treatment with the TGFβ inhibitor PF-60952229 to mice previously receiving the CDK4 / 6 inhibitor palbociclib or palbociclib + fulvestrant for 21 days was shown, 66 days after the start of treatment. It is a figure which shows that inhibition of tumor growth tends to increase in MCF7 ER ⁺ xenograft breast cancer tumor model. MCF7 ER ⁺ xenograft breast cancer tumor after 21 days of administration of the TGFβ inhibitor PF-60952229 and the CDK4 / 6 inhibitor palbociclib (PD-0332991) or the combination of palbociclib + fulvestrant for 21 days. It is a figure which shows that the inhibition of pSMAD2 is improved in a model. When a combination of the TGFβ inhibitor PF-60952229 and the CDK4 / 6 inhibitor palbociclib (PD-0332991) + fulvestrant was administered for 21 days, pS807 / 811 Rb in the MCF7 ER ⁺ xenograft breast cancer tumor model. It is a figure which shows that the inhibition of is improved.

The invention can be more easily understood by reference to the following detailed description of preferred embodiments of the invention and examples contained herein. It should be understood that the terminology used herein is intended only to describe specific embodiments and is not intended to be limiting. Further, it should be understood that the terminology used herein has its usual meaning known in the relevant field, unless specifically defined herein.

As used herein, the singular forms "one (a)", "one (an)", and "the" include plural references unless otherwise indicated. For example, an "an" excipient comprises one or more excipients.

As used herein, the term "about", when used to modify a parameter defined by a number (eg, the dose of a TGFβ inhibitor or CDK inhibitor), is that parameter. Means that it can vary to 10% below or above the number mentioned for. For example, a dose of about 5 mg can vary between 4.5 mg and 5.5 mg.

As used herein, "acting agents," "components," "compositions," "compounds," "substances," "targeting agents," "targeted therapeutic agents," and "therapeutic agents." The term "drug" is used to refer to a compound of the invention, specifically a TGFβ inhibitor and a CDK inhibitor. Can be used without distinction.

The following abbreviations may be used herein: DMSO (dimethyl sulfoxide), FBS (fetal bovine serum), RPMI (Roswell Park Memorial Institute medium), mpk (mg / kg of drug per kg body weight of animal) or mg), and w / w (weight per weight).

Cyclin-dependent kinases (CDKs) and related serine / threonine kinases are important cellular enzymes that play an essential role in the regulation of cell division and proliferation. CDK inhibitors include Pan-CDK inhibitors that target a wide range of CDKs, or selective CDK inhibitors that target specific CDKs. In addition to CDK, CDK inhibitors have activity against targets such as Aurora A, Aurora B, Chk1, Chk2, ERK1, ERK2, GST-ERK1, GSK-3α, GSK-3β, PDGFR, TrkA, and VEGFR. obtain. CDK inhibitors include, but are not limited to, abemaciclib, alvocidib, dinasiclib, palbociclib, ribociclib, trilaciclib, lelocyclib, roscobitin, AT7519, AZD5438, BMS-265246, BMS-387032, BS-1811, JNJ-7706621, -8776, P276-00, PHA-793887, R547, RO-3306, and SU9516 are included. Examples of Pan-CDK inhibitors include, but are not limited to, alvocidib, dynacyclib, roscobitin, AT7519, AZD5438, BMS-387032, P276-00, PHA-793887, R547, and SU9516. A non-limiting example of a CDK1 inhibitor is RO-3306. Examples of CDK2 inhibitors include, but are not limited to, K03861 and MK-8776. Examples of CDK1 / 2 inhibitors include, but are not limited to, BMS-265246 and JNJ-7706621. Examples of CDK4 / 6 inhibitors include, but are not limited to, abemaciclib, ribociclib, and palbociclib. A non-limiting example of a CDK7 inhibitor is BS-181.

In one embodiment, the CDK4 / 6 inhibitor of the present invention includes palbociclib. Unless otherwise indicated herein, palbociclib (also referred to herein as "palbo" or "parbo") is 6-acetyl-8-cyclopentyl-5-methyl-2- (also referred to herein). 5-Piperazine-1-yl-pyridine-2-ylamino) -8H-pyrido [2,3-d] Refers to pyrimidine-7-one or a pharmaceutically acceptable salt thereof.

Some embodiments relate to pharmaceutically acceptable salts of the compounds described herein. Pharmaceutically acceptable salts of the compounds described herein include acid and base salts thereof.

Some embodiments also relate to pharmaceutically acceptable acid addition salts of the compounds described herein. Suitable acid addition salts are formed from acids that form non-toxic salts. Non-limiting examples of suitable acid addition salts, ie, salts containing pharmacologically acceptable anions, are, but are not limited to, acetates, citrates, adipates, asparagitates, benzoic acids. Salts, besilates, bicarbonates / carbonates, bicarbonates / sulfates, tartrates, borates, cansilates, citrates, cyclamates, edicates, esylates, ethanesulfones. Acids, grate, fumarate, gluceptate, gluconate, glucronate, hexafluorophosphate, hibenzate, hydrochloride / chloride salt, hydrobromide / bromide salt, iodide Hydrochloride / iodide salt, isethionate, lactate, malate, maleate, malonate, mesylate, methanesulfonate, methylsulfate, naphthylate, 2-napsylate, Nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate Includes acid salts, tannates, tartrates, p-toluenesulfonates, tosylates, trifluoroacetates, and xinafoates.

Further embodiments relate to base addition salts of the compounds described herein. Suitable base addition salts are formed from bases that form non-toxic salts. Non-limiting examples of suitable basic salts include salts of aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc. Is done.

The compounds described herein, which are essentially basic, can form a wide range of salts with various inorganic and organic acids. The acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds described herein are those that form nontoxic acid addition salts, eg, pharmacologically acceptable. Salts containing anions that can be formed, such as hydrochlorides, hydrobromide, hydroiodide, nitrates, sulfates, bisulfates, phosphates, acidic phosphates, isonicotinates, acetates, Lactate, salicylate, citrate, acidic citrate, tartrate, pantothenate, heavy tartrate, ascorbate, succinate, maleate, gentisinate, fumarate, glucon Acids, glucurates, saccharates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pamoates [ie, 1,1'-methylene-bis- (2-hydroxy-3-naphthoate)]. The compounds described herein, including basic moieties such as amino groups, can form pharmaceutically acceptable salts with various amino acids in addition to the acids described above.

Chemical bases that can be used as reagents to prepare pharmaceutically acceptable basic salts of the compounds described herein that are inherently acidic form non-toxic basic salts with such compounds. It is a thing. Such non-toxic basic salts include, but are not limited to, such pharmacologically such as alkali metal cations (eg, potassium and sodium) and alkaline earth metal cations (eg, calcium and magnesium). Includes acceptable cations, ammonium or water soluble amine addition salts such as N-methylglucamine- (meglumin), and lower alkanolammoniums, as well as those derived from other basic salts of pharmaceutically acceptable organic amines. ..

Hemi salts of acids and bases, such as hemi sulphates and hemi calcium salts, can also be formed.

See Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002) by Stahl and Wermut for a summary of suitable salts. Methods for making pharmaceutically acceptable salts of the compounds described herein are known to those of skill in the art.

The term "solvate" is used herein to describe a molecular complex comprising the compounds described herein and one or more pharmaceutically acceptable solvent molecules such as water and ethanol. Will be done.

The compounds described herein can also be present in non-solvate and solvate forms. Accordingly, some embodiments relate to hydrates and solvates of the compounds described herein.

The compounds described herein, including one or more asymmetric carbon atoms, can exist as more than one stereoisomer. Geometric cis / trans (or Z / E) isomers are possible if the compounds described herein contain alkenyl or alkenylene groups. If structural isomers are intercompatible through a low energy barrier, tautomeric isomerism (“tautomerization”) can occur. This may take the form of proton tautomerism in the compounds described herein, including, for example, iminos, ketos, or oxime groups, or so-called valence tautomers in compounds containing aromatic moieties. Can take form. A single compound can exhibit more than one type of isomerism.

The compounds of the embodiments described herein include all stereoisomers (eg, cis and trans isomers) and all optical isomers (eg, R and S enantiomers) of the compounds described herein. ), As well as racemic mixtures of such isomers, diastereomeric mixtures, and other mixtures. Although all stereoisomers are included in the claims of the present invention, those skilled in the art will recognize that certain stereoisomers may be preferred.

In some embodiments, the compounds described herein may be present in several tautomeric forms, including enol and imine forms and keto and enamine forms, as well as geometric isomers, and mixtures thereof. All such tautomeric forms are included within the scope of this embodiment. Tautomers are present in solution as a mixture of tautomer sets. In solid form, one tautomer usually predominates. Even though one tautomer is described, embodiments of the invention include all tautomers of the compounds of the invention.

Within the scope of this embodiment, all stereoisomers and geometric isomers of the compounds described herein, including compounds exhibiting two or more types of isomers, and mixtures consisting of one or more thereof. , And tautomeric types are included. Also included are acid addition or basic salts whose counterions are optically active, such as d-lactate or l-lysine or racemic, such as dl-tartrate or dl-arginine.

The present embodiment also includes the atropisomers of the compounds described herein. Atropisomers refer to compounds that can be separated into rotation-restricted isomers.

The cis / trans isomers can be separated by conventional techniques well known to those of skill in the art, such as chromatography and fractional crystallization.

Conventional techniques for preparing / isolating individual enantiomers include chiral synthesis from suitable optically pure precursors, or racemates (or salts using, for example, chiral high performance liquid chromatography (HPLC)). Or the racemic form of the derivative) is included.

Alternatively, the racemate (or racemic precursor) may be a suitable optically active compound, such as an alcohol, or 1-phenylethylamine in cases where the compounds described herein contain an acidic or basic moiety. Alternatively, it can be reacted with a base or acid such as tartaric acid. The resulting diastereomeric mixture can be separated by chromatography and / or fractional crystallization, and one or both of the diastereoisomers are converted to the corresponding pure enantiomers by means well known to those of skill in the art.

As used herein, the term "treat" refers to a disorder or condition to which such a term applies, or one or more symptoms of such a disorder or condition, unless otherwise indicated. It means to recover, to alleviate, to impede its progress, or to prevent it. As used herein, the term "treatment" refers to the act of treatment, unless otherwise indicated, where "treatment" is as defined immediately before.

"Patients" treated according to the present invention include, but are not limited to, any homeothermic animal such as humans, monkeys, or other lower primates, horses, dogs, rabbits, guinea pigs, or mice. For example, the patient is human. Those skilled in the medical field can easily identify individual patients with breast cancer, especially HR-positive, HER2-negative, advanced, or metastatic breast cancer, and individual patients in need of treatment. can do.

The term "advanced" as used herein includes locally advanced (non-metastatic) and metastatic diseases when it is associated with breast cancer. Locally advanced breast cancer that can or cannot be treated for curative purposes, and metastatic diseases that cannot be treated for curative purposes are included within the scope of "advanced breast cancer" used in the present invention. Those skilled in the art will be able to recognize and diagnose advanced breast cancer in patients.

The "duration of response" for the purposes of the present invention means from the time of recording the inhibition of growth of the tumor model by drug treatment to the time of returning to a growth rate similar to the growth rate before treatment.

The term "additive" is used to mean that the result of a combination of two compounds, components, or target drug is not greater than the sum of each compound, component, or individual target drug. .. The term "additive" means that there is no improvement in the disease state or disorder to be treated as compared to the individual use of each compound, component, or target drug.

The term "synergistic" or "synergistic" is used to mean that the result of a combination of two compounds, constituents, or targeting agents is better than the combined sum of each agonist. .. The term "synergistic" or "synergistic" means that the disease state or disorder to be treated is ameliorated over the individual use of each compound, component, or target drug. This improvement in the disease state or disorder being treated is a "synergistic effect." A "synergistic amount" is the amount of a combination of two compounds, constituents, or targeting agents that results in a synergistic effect, where "synergistic" is as defined herein.

To determine the synergistic interaction between one or two components, the components are administered to patients in need of treatment over various w / w ratio ranges and doses of effect. The optimal range for, and the absolute dose range of each component for effect can be definitively measured. However, observation of synergistic effects in in vitro or in vitro models can predict effects in humans and other species, and in vitro or in vitro models measure synergistic effects as described herein. The results of such studies also exist for, by applying pharmacokinetic / pharmacodynamic methods, the range of effective dose and plasma concentration ratios required in humans and other species as well as It can be used to predict absolute dose and plasma concentration.

According to the invention, an amount of the first compound or component was combined with an amount of the second compound or component, and the combined amount was advanced for breast cancer, especially HR positive, HER2-negative. , Or is effective in the treatment of metastatic breast cancer. Combined, the effective amount will alleviate to some extent one or more of the symptoms of the disorder being treated. For cancer treatment, effective amounts (1) reduce the size of the tumor, (2) inhibit the appearance of tumor metastases (ie, slow down to some extent, preferably stop), (3) grow the tumor. Alternatively, it inhibits the invasiveness of the tumor to some extent (ie, slows it down to some extent, preferably stops it), and / or (4) alleviates (or preferably eliminates) one or more signs or symptoms associated with the cancer to some extent. ) Refers to the amount that has an effect. The therapeutic or pharmacological efficacy of dose and dosing regimens is also characterized as the ability to induce, enhance, maintain, or prolong disease control and / or overall survival in patients with these specific tumors. It can be attached, which can be measured as an extension of the time it takes for the disease to progress.

In one embodiment, the invention is an effective amount of a CDK inhibitor in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer, in patients in need thereof. With respect to methods for treating breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, comprising administering a certain amount of TGFβ inhibitor in combination with. In a further embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and an amount of a CDK inhibitor, breast cancer, particularly HR-positive, HER2-negative, advanced. A method for treating breast cancer that has or is metastatic, and the combined dose is effective in treating breast cancer, especially HR-positive, HER2-negative, advanced, or metastatic breast cancer. Regarding the method. In another embodiment, the invention relates to a combination of a TGFβ inhibitor and a CDK inhibitor for use in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. In another embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and an amount of a CDK inhibitor, especially for breast cancer, particularly HR-positive, HER2-negative. A method for treating advanced or metastatic breast cancer, the combined dose achieving synergistic effects in the treatment of breast cancer, especially HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding how to do it. In another embodiment, the invention relates to a synergistic combination of a TGFβ inhibitor and a CDK inhibitor for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. In one embodiment, the method or use of the invention relates to a synergistic combination of a targeted therapeutic agent, specifically a TGFβ inhibitor and a CDK inhibitor.

In one embodiment, the invention is an effective amount of a CDK inhibitor in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer, in patients in need thereof. With respect to methods for treating breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, comprising administering a certain amount of TGFβ inhibitor in combination with. In a further embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and an amount of a CDK inhibitor, breast cancer, particularly HR-positive, HER2-negative, advanced. A method for treating breast cancer that has or is metastatic, and the combined dose is effective in treating breast cancer, especially HR-positive, HER2-negative, advanced, or metastatic breast cancer. Regarding the method. In another embodiment, the invention relates to a combination of a TGFβ inhibitor and a CDK inhibitor for use in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. In another embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and an amount of a CDK inhibitor, especially for breast cancer, particularly HR-positive, HER2-negative. A method for treating advanced or metastatic breast cancer, the combined dose achieving synergistic effects in the treatment of breast cancer, especially HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding how to do it. In another embodiment, the invention relates to a synergistic combination of a TGFβ inhibitor and a CDK inhibitor for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. In one embodiment, the method or use of the invention relates to a synergistic combination of a targeted therapeutic agent, specifically a TGFβ inhibitor and a CDK inhibitor.

In one embodiment, the invention is an amount of CDK4 / 6 that is effective in treating breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer, in patients in need thereof. It relates to a method for treating breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, which comprises administering a certain amount of TGFβ inhibitor in combination with the inhibitor. In a further embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and an amount of a CDK4 / 6 inhibitor for breast cancer, particularly HR positive, HER2-negative. A method for treating advanced or metastatic breast cancer, the combined dose of which is effective in treating breast cancer, especially HR-positive, HER2-negative, advanced or metastatic breast cancer. There is a method. In another embodiment, the invention relates to a combination of a TGFβ inhibitor and a CDK4 / 6 inhibitor in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. In another embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and an amount of a CDK4 / 6 inhibitor for breast cancer, particularly HR positive, HER2 negative. A method for treating advanced or metastatic breast cancer, the combined dose of which is synergistic in the treatment of breast cancer, especially HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding how to achieve. In another embodiment, the invention is synergistic of TGFβ and CDK4 / 6 inhibitors for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding the combination. In one embodiment, the method or use of the invention relates to a synergistic combination of a targeted therapeutic agent, specifically a TGFβ inhibitor and a CDK4 / 6 inhibitor.

In one embodiment, the invention is an amount of CDK4 / 6 that is effective in treating breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer, in patients in need thereof. It relates to a method for treating breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, which comprises administering a certain amount of TGFβ inhibitor in combination with the inhibitor. In a further embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and an amount of a CDK4 / 6 inhibitor for breast cancer, particularly HR positive, HER2-negative. A method for treating advanced or metastatic breast cancer, the combined dose of which is effective in treating breast cancer, especially HR-positive, HER2-negative, advanced or metastatic breast cancer. There is a method. In another embodiment, the invention relates to a combination of a TGFβ inhibitor and a CDK4 / 6 inhibitor in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. In another embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and an amount of a CDK4 / 6 inhibitor for breast cancer, particularly HR positive, HER2 negative. A method for treating advanced or metastatic breast cancer, the combined dose of which is synergistic in the treatment of breast cancer, especially HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding how to achieve. In another embodiment, the invention is synergistic of TGFβ and CDK4 / 6 inhibitors for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding the combination. In one embodiment, the method or use of the invention relates to a synergistic combination of a targeted therapeutic agent, specifically a TGFβ inhibitor and a CDK4 / 6 inhibitor.

In one embodiment, the invention is an amount of palbociclib or an amount thereof that is effective in treating breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer, in patients in need thereof. A certain amount of 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropane-2-) combined with a pharmaceutically acceptable salt Il) Methods for treating breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, comprising administration of nicotine amide (PF-60952229) or a pharmaceutically acceptable salt thereof. Regarding. In a further embodiment, the invention presents an amount of 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1) in a patient in need thereof. , 3-Dihydroxypropan-2-yl) nicotine amide (PF-60952229) or a pharmaceutically acceptable salt thereof, and a certain amount of palbociclib or a pharmaceutically acceptable salt thereof, including administration of breast cancer, particularly A method for treating HR-positive, HER2-negative, advanced or metastatic breast cancer, the combined amount of which is breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic. Concerning methods that are effective in the treatment of breast cancer. In another embodiment, the invention is 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropane-2-yl). A combination of nicotinamide (PF-60952229) or a pharmaceutically acceptable salt thereof and palbociclib or a pharmaceutically acceptable salt thereof, the combined amount of which is breast cancer, particularly HR positive, HER2-negative. Concerning combinations that are effective in the treatment of advanced or metastatic breast cancer. In another embodiment, the invention presents an amount of 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- ( Breast cancer, particularly including administration of 1,3-dihydroxypropan-2-yl) nicotine amide (PF-60952229) or a pharmaceutically acceptable salt thereof, and an amount of palbociclib or a pharmaceutically acceptable salt thereof. , HR-positive, HER2-negative, advanced or metastatic breast cancer, the combined amount of which is breast cancer, especially HR-positive, HER2-negative, advanced or metastatic. On how to achieve synergistic effects in the treatment of breast cancer. In another embodiment, the invention is for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer, 4- (2- (5-chloro-2-fluorophenyl). ) -5-Isopropylpyridin-4-ylamino) -N- (1,3-dihydroxypropan-2-yl) nicotinamide (PF-609522229) or a pharmaceutically acceptable salt thereof and palbociclib or a pharmaceutically acceptable salt thereof. Concerning a synergistic combination with the resulting salt. In one embodiment, the methods or uses of the invention are targeted therapeutic agents, specifically 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- ( 1,3-Dihydroxypropan-2-yl) Nicotinamide (PF-60952229) or a pharmaceutically acceptable salt thereof and a synergistic combination of palbociclib or a pharmaceutically acceptable salt thereof.

"Standard clinical dosing regimen" as used herein refers to a regimen for administering a substance, agent, compound, or composition typically used in the clinical setting. A "standard clinical dosing regimen" includes a "standard clinical dose" or a "standard dosing schedule".

A "non-standard clinical dosing regimen", when used herein, administers a substance, agent, compound, or composition that differs from the amount, dose, or schedule typically used in the clinical setting. Refers to the regimen for. A "non-standard clinical dosing regimen" includes a "non-standard clinical dose" or a "non-standard dosing schedule".

In one embodiment, the invention is an effective amount of a CDK inhibitor in the treatment of breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, in patients in need thereof. A method for treating breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer, comprising administering a certain amount of a TGFβ inhibitor in combination with a CDK inhibitor. , Concerning how to be administered according to a non-standard clinical dosing regimen. In a further embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and an amount of a CDK inhibitor, breast cancer, particularly HR-positive, HER2-negative, advanced. A method for treating breast or metastatic breast cancer in which a CDK inhibitor is administered according to a non-standard clinical dosing regimen, and the combined dose is breast cancer, especially HR positive, HER2-negative. With respect to methods that are effective in the treatment of advanced or metastatic breast cancer. In another embodiment, the invention is a combination of a TGFβ inhibitor and an amount of a CDK inhibitor for use in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. It relates to a combination in which the CDK inhibitor is administered according to a non-standard clinical dosing regimen. In another embodiment, the invention comprises administering a certain amount of a TGFβ inhibitor and a certain amount of a CDK inhibitor to a patient in need thereof for breast cancer, particularly HR positive, HER2-negative. A method for treating advanced or metastatic breast cancer in which a CDK inhibitor is administered according to a non-standard clinical dosing regimen, and the combined dose is for breast cancer, especially HR positive, HER2-negative. On how to achieve synergistic effects in the treatment of advanced or metastatic breast cancer. In another embodiment, the invention is a synergistic combination of TGFβ and CDK inhibitors for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. The CDK inhibitor is administered according to a non-standard clinical dosing regimen, relating to a combination.

In one embodiment, the invention is an effective amount of a CDK inhibitor in the treatment of breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, in patients in need thereof. A method for treating breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer, comprising administering a certain amount of a TGFβ inhibitor in combination with a CDK inhibitor. , Concerning how to be administered according to a non-standard clinical dosing regimen. In a further embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and an amount of a CDK inhibitor, breast cancer, particularly HR-positive, HER2-negative, advanced. A method for treating breast or metastatic breast cancer in which a CDK inhibitor is administered according to a non-standard clinical dosing regimen, and the combined dose is breast cancer, especially HR positive, HER2-negative. With respect to methods that are effective in the treatment of advanced or metastatic breast cancer. In another embodiment, the invention is the use of a combination of a TGFβ inhibitor and a CDK inhibitor in the treatment of breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, the CDK. For use in which the inhibitor is administered according to a non-standard clinical dosing regimen. In another embodiment, the invention comprises administering a certain amount of a TGFβ inhibitor and a certain amount of a CDK inhibitor to a patient in need thereof for breast cancer, particularly HR positive, HER2-negative. A method for treating advanced or metastatic breast cancer in which a CDK inhibitor is administered according to a non-standard clinical dosing regimen, and the combined dose is for breast cancer, especially HR positive, HER2-negative. On how to achieve synergistic effects in the treatment of advanced or metastatic breast cancer. In another embodiment, the invention uses a combination of a certain amount of a TGFβ inhibitor and a CDK inhibitor for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. It relates to the use in which a CDK inhibitor is administered according to a non-standard clinical dosing regimen.

In one embodiment, the invention is an amount of CDK4 / 6 that is effective in treating breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer, in patients in need thereof. A method for treating breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer, comprising administering a certain amount of TGFβ inhibitor in combination with the inhibitor, CDK4 /. 6 The present invention relates to a method in which an inhibitor is administered according to a non-standard clinical dosing regimen. In a further embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and an amount of a CDK4 / 6 inhibitor for breast cancer, particularly HR positive, HER2-negative. A method for treating advanced or metastatic breast cancer in which a CDK4 / 6 inhibitor is administered according to a non-standard clinical dosing regimen, and the combined amount is breast cancer, especially HR positive. , HER2-negative, effective in the treatment of advanced or metastatic breast cancer. In another embodiment, the invention is a combination of a TGFβ inhibitor and a CDK4 / 6 inhibitor in the treatment of breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, CDK4. / 6 Inhibitors are administered according to a non-standard clinical dosing regimen, relating to a combination. In another embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and an amount of a CDK4 / 6 inhibitor for breast cancer, particularly HR positive, HER2 negative. A method for treating advanced or metastatic breast cancer, in which a CDK4 / 6 inhibitor is administered according to a non-standard clinical dosing regimen, and the combined dose is breast cancer, especially HR positive. HER2-negative, advanced or metastatic breast cancer treatment to achieve synergistic effects. In another embodiment, the invention is a synergistic combination of a TGFβ inhibitor and a CDK4 / 6 inhibitor for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. With respect to the combination, wherein the CDK4 / 6 inhibitor is administered according to a non-standard clinical dosing regimen.

In one embodiment, the invention is an amount of CDK4 / 6 that is effective in treating breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer, in patients in need thereof. A method for treating breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer, comprising administering a certain amount of TGFβ inhibitor in combination with the inhibitor, CDK4 /. 6 The present invention relates to a method in which an inhibitor is administered according to a non-standard clinical dosing regimen. In a further embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and an amount of a CDK4 / 6 inhibitor for breast cancer, particularly HR positive, HER2-negative. A method for treating advanced or metastatic breast cancer in which a CDK4 / 6 inhibitor is administered according to a non-standard clinical dosing regimen, and the combined amount is breast cancer, especially HR positive. , HER2-negative, effective in the treatment of advanced or metastatic breast cancer. In another embodiment, the invention is a TGFβ inhibitor and an amount of a CDK4 / 6 inhibitor for use in the treatment of breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer. The CDK4 / 6 inhibitor is administered according to a non-standard clinical dosing regimen. In another embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and an amount of a CDK4 / 6 inhibitor for breast cancer, particularly HR positive, HER2 negative. A method for treating advanced or metastatic breast cancer, in which a CDK4 / 6 inhibitor is administered according to a non-standard clinical dosing regimen, and the combined dose is breast cancer, especially HR positive. HER2-negative, advanced or metastatic breast cancer treatment to achieve synergistic effects. In another embodiment, the invention is a synergistic combination of a TGFβ inhibitor and a CDK4 / 6 inhibitor for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. With respect to the combination, wherein the CDK4 / 6 inhibitor is administered according to a non-standard clinical dosing regimen.

In one embodiment, the invention is an amount of palbociclib or an amount thereof that is effective in treating breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer, in patients in need thereof. A certain amount of 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropane-2-) combined with a pharmaceutically acceptable salt Il) Methods for treating breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, comprising administering nicotine amide (PF-60952229) or a pharmaceutically acceptable salt thereof. It relates to a method in which palbociclib or a pharmaceutically acceptable salt thereof is administered according to a non-standard clinical dosing regimen. In a further embodiment, the invention presents an amount of 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1) in a patient in need thereof. , 3-Dihydroxypropan-2-yl) nicotine amide (PF-60952229) or a pharmaceutically acceptable salt thereof, and a certain amount of palbociclib or a pharmaceutically acceptable salt thereof, including administration of breast cancer, particularly A method for treating HR-positive, HER2-negative, advanced or metastatic breast cancer in which palbociclib or a pharmaceutically acceptable salt thereof is administered according to a non-standard clinical dosing regimen and further combined. The amount relates to a method that is effective in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. In another embodiment, the invention is 4- (2- (5-chloro-2-) for use in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. Fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropan-2-yl) nicotinamide (PF-609522229) or a pharmaceutically acceptable salt thereof and palbociclib or its pharmaceuticals. Consistent with a combination of palbociclib or a pharmaceutically acceptable salt thereof, which is an acceptable combination with a non-standard clinical dosing regimen. In another embodiment, the invention presents an amount of 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N-( Breast cancer, particularly including administration of 1,3-dihydroxypropan-2-yl) nicotine amide (PF-60952229) or a pharmaceutically acceptable salt thereof, and an amount of palbociclib or a pharmaceutically acceptable salt thereof. Palbociclib or a pharmaceutically acceptable salt thereof is administered according to a non-standard clinical dosing regimen and is a method for treating HR-positive, HER2-negative, advanced or metastatic breast cancer. The combined amounts relate to how to achieve synergistic effects in the treatment of breast cancer, especially HR-positive, HER2-negative, advanced or metastatic breast cancer. In another embodiment, the invention is for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer, 4- (2- (5-chloro-2-fluorophenyl). ) -5-Isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropan-2-yl) nicotinamide (PF-609522229) or a pharmaceutically acceptable salt thereof and palbociclib or a pharmaceutically acceptable salt thereof. A synergistic combination with a possible salt, wherein palbociclib or a pharmaceutically acceptable salt thereof is administered according to a non-standard clinical dosing regimen.

"Low dose" as used herein refers to an amount or dose of a substance, agent, compound, or composition that is less than the amount or dose typically used in clinical practice.

In one embodiment, the invention is a low dose CDK inhibitor effective in treating breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, in patients in need thereof. With respect to methods for treating breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, comprising administering a certain amount of TGFβ inhibitor in combination with. In a further embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and a low dose of a CDK inhibitor, breast cancer, particularly HR positive, HER2-negative, advanced. A method for treating breast cancer that has or is metastatic, and the combined dose is effective in treating breast cancer, especially HR-positive, HER2-negative, advanced, or metastatic breast cancer. Regarding the method. In another embodiment, the invention is a combination of a TGFβ inhibitor and a low dose CDK inhibitor for use in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding. In another embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and a low dose of a CDK inhibitor for breast cancer, particularly HR positive, HER2-negative. A method for treating advanced or metastatic breast cancer, the combined dose achieving synergistic effects in the treatment of breast cancer, especially HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding how to do it. In another embodiment, the invention is synergistic of a TGFβ inhibitor and a low dose CDK inhibitor for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding the combination.

In one embodiment, the invention is a low dose CDK inhibitor effective in treating breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, in patients in need thereof. With respect to methods for treating breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, comprising administering a certain amount of TGFβ inhibitor in combination with. In a further embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and a low dose of a CDK inhibitor, breast cancer, particularly HR positive, HER2-negative, advanced. A method for treating breast cancer that has or is metastatic, and the combined dose is effective in treating breast cancer, especially HR-positive, HER2-negative, advanced, or metastatic breast cancer. Regarding the method. In another embodiment, the invention is a combination of a TGFβ inhibitor and a low dose CDK inhibitor for use in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding. In another embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and a low dose of a CDK inhibitor for breast cancer, particularly HR positive, HER2-negative. A method for treating advanced or metastatic breast cancer, the combined dose achieving synergistic effects in the treatment of breast cancer, especially HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding how to do it. In another embodiment, the invention is synergistic of a TGFβ inhibitor and a low dose CDK inhibitor for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding the combination.

In one embodiment, the invention is a low dose CDK4 / 6 effective in treating breast cancer, especially HR-positive, HER2-negative, advanced or metastatic breast cancer, in patients in need thereof. It relates to a method for treating breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, which comprises administering a certain amount of TGFβ inhibitor in combination with the inhibitor. In a further embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and a low dose of a CDK4 / 6 inhibitor for breast cancer, particularly HR positive, HER2-negative. A method for treating advanced or metastatic breast cancer, the combined dose of which is effective in treating breast cancer, especially HR-positive, HER2-negative, advanced or metastatic breast cancer. There is a method. In another embodiment, the invention is a TGFβ inhibitor and a low dose CDK4 / 6 inhibitor for use in the treatment of breast cancer, in particular HR positive, HER2-negative, advanced or metastatic breast cancer. Regarding the combination of. In another embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and a low dose of a CDK4 / 6 inhibitor for breast cancer, particularly HR positive, HER2 negative. A method for treating advanced or metastatic breast cancer, the combined dose of which is synergistic in the treatment of breast cancer, especially HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding how to achieve. In another embodiment, the invention is a synergistic combination of a TGFβ inhibitor and a low dose CDK4 / 6 inhibitor for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. Combination.

In one embodiment, the invention is a low dose CDK4 / 6 effective in treating breast cancer, especially HR-positive, HER2-negative, advanced or metastatic breast cancer, in patients in need thereof. It relates to a method for treating breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, which comprises administering a certain amount of TGFβ inhibitor in combination with the inhibitor. In a further embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and a low dose of a CDK4 / 6 inhibitor for breast cancer, particularly HR positive, HER2-negative. A method for treating advanced or metastatic breast cancer, the combined dose of which is effective in treating breast cancer, especially HR-positive, HER2-negative, advanced or metastatic breast cancer. There is a method. In another embodiment, the invention is a TGFβ inhibitor and a low dose CDK4 / 6 inhibitor for use in the treatment of breast cancer, in particular HR positive, HER2-negative, advanced or metastatic breast cancer. Regarding the combination of. In another embodiment, the invention comprises administering to a patient in need thereof an amount of a TGFβ inhibitor and a low dose of a CDK4 / 6 inhibitor for breast cancer, particularly HR positive, HER2 negative. A method for treating advanced or metastatic breast cancer, the combined dose of which is synergistic in the treatment of breast cancer, especially HR-positive, HER2-negative, advanced or metastatic breast cancer. Regarding how to achieve. In another embodiment, the invention is a synergistic combination of a TGFβ inhibitor and a low dose CDK4 / 6 inhibitor for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. Combination.

In one embodiment, the invention is a low dose of palbociclib or a dose thereof that is effective in treating breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer, in patients in need thereof. A certain amount of 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropane-2) in combination with a pharmaceutically acceptable salt -Il) For treating breast cancer, particularly HR-positive, HER2-negative, advanced or metastatic breast cancer, comprising administration of nicotine amide (PF-60952229) or a pharmaceutically acceptable salt thereof. Regarding the method. In a further embodiment, the invention presents an amount of 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1) in a patient in need thereof. , 3-Dihydroxypropan-2-yl) nicotine amide (PF-60952229) or a pharmaceutically acceptable salt thereof, and low doses of palbociclib or a pharmaceutically acceptable salt thereof, including administration of breast cancer, particularly A method for treating HR-positive, HER2-negative, advanced or metastatic breast cancer, the combined dose of which is breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic. Concerning methods that are effective in the treatment of breast cancer. In another embodiment, the invention is 4- (2- (5-chloro-2-) for use in the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. Fluorophenyl) -5-isopropylpyridin-4-ylamino) -N- (1,3-dihydroxypropan-2-yl) nicotinamide (PF-609522229) or a pharmaceutically acceptable salt thereof and low doses of palbociclib or With respect to its combination with a pharmaceutically acceptable salt. In another embodiment, the invention presents an amount of 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- ( Breast cancer, particularly including administration of 1,3-dihydroxypropan-2-yl) nicotine amide (PF-60952229) or a pharmaceutically acceptable salt thereof, and low doses of palbociclib or a pharmaceutically acceptable salt thereof. , HR-positive, HER2-negative, advanced or metastatic breast cancer, the combined amount of which is breast cancer, especially HR-positive, HER2-negative, advanced or metastatic. On how to achieve synergistic effects in the treatment of breast cancer. In another embodiment, the invention relates to an amount of 4- (2- (5-chloro-2)) for the treatment of breast cancer, in particular HR-positive, HER2-negative, advanced or metastatic breast cancer. -Fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropan-2-yl) nicotinamide (PF-609522229) or a pharmaceutically acceptable salt thereof and a low dose of palbociclib. Or with respect to its synergistic combination with a pharmaceutically acceptable salt.

Appropriate amounts, doses, or dosages of each compound for use in the combination of the invention for administration to a patient according to known methods, age, body weight, overall health, administration. Factors such as the compound to be administered, the route of administration, the nature and degree of progression of HR-positive, HER2-negative, advanced or metastatic breast cancer, and the presence of other drug treatments, in particular. Let's decide.

In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is about 125 mg once daily, about 100 mg once daily, about 75 mg once daily, or about 50 mg daily. It is administered in a single daily dose. In one embodiment, which is the recommended starting dose or standard clinical dose, palbociclib or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 125 mg once daily. In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered at a non-standard clinical dose. In one embodiment, the non-standard clinical dose is a low dose of palbociclib or a pharmaceutically acceptable salt thereof. For example, palbociclib or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg once daily, about 75 mg once daily, or about 50 mg once daily. In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 100 mg. In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 75 mg. In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 50 mg. The dosage provided herein refers to the dose of the free base form of palbociclib or is calculated as the free base equivalent of the palbociclib salt form administered. A dosage or amount of palbociclib, such as 100 mg, 75 mg, or 50 mg, refers to a free base equivalent. This dosage regimen can be adjusted to provide the optimal therapeutic response. For example, the dose can be proportionally reduced or increased, as indicated by the urgency of the treatment situation.

In one embodiment, PF-06783600 or a pharmaceutically acceptable salt thereof is about 125 mg once daily, about 100 mg once daily, about 75 mg once daily, or about 50 mg 1 It is administered at a daily dosage of once daily. In one embodiment, PF-06783600 or a pharmaceutically acceptable salt thereof is administered in a daily dosage of about 125 mg once daily. In one embodiment, PF-06783600 or a pharmaceutically acceptable salt thereof is administered at a non-standard clinical dose. In one embodiment, the non-standard clinical dose is a low dose of PF-06673600 or a pharmaceutically acceptable salt thereof. For example, PF-06673600 or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg once daily, about 75 mg once daily, or about 50 mg once daily. In one embodiment, PF-06783600 or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 100 mg. In one embodiment, PF-06783600 or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 75 mg. In one embodiment, PF-06783600 or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 50 mg. The dosage provided herein refers to the dose in the free base form of PF-06873600 or is calculated as the free base equivalent of the PF-06673600 salt form administered. For example, a dosage or amount of PF-06783600, such as 100 mg, 75 mg, or 50 mg, refers to a free base equivalent. This dosage regimen can be adjusted to provide the optimal therapeutic response. For example, the dose can be proportionally reduced or increased, as indicated by the urgency of the treatment situation.

Implementation of the methods of the invention can be achieved through a variety of dosing regimens or dosing regimens. The compounds of the combination of the invention can be administered intermittently, simultaneously or continuously. In one embodiment, the compounds of the combinations of the invention can be administered in a co-dosing regimen.

The dosing regimen or dosing regimen can be repeated as needed to achieve the desired reduction or reduction of cancer cells. A "continuous dosing schedule", as used herein, is a dosing regimen or dosing regimen without interruption of dosing, eg, without days of no treatment. Repeating a 21- or 28-day treatment cycle with no dosing interruption during the treatment cycle is an example of a continuous dosing schedule. In one embodiment, the combination of compounds of the invention can be administered on a continuous dosing schedule. In one embodiment, the compounds of the combination of the invention can be administered simultaneously on a continuous dosing schedule.

In one embodiment, PF-60952229 or a pharmaceutically acceptable salt thereof is administered once daily to include a full 28-day cycle. Repeating the 28-day cycle is continued during the treatment with the combination of the invention.

In one embodiment, PF-60952229 or a pharmaceutically acceptable salt thereof is administered once daily to include a full cycle of 21 days. Repeating the 21-day cycle is continued during the treatment with the combination of the invention.

A standard recommended dosing regimen, including a standard dosing schedule for palbociclib or a pharmaceutically acceptable salt thereof, is given once daily for 21 consecutive days, followed by no treatment for 7 days, 28. Include the entire day cycle. Repeating the 28-day cycle is continued during the treatment with the combination of the invention.

In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered under a non-standard dosing schedule. For example, palbociclib or a pharmaceutically acceptable salt thereof should be administered once daily to include a full 28-day cycle. Repeating the 28-day cycle is continued during the treatment with the combination of the invention.

In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered under a non-standard dosing schedule. For example, palbociclib or a pharmaceutically acceptable salt thereof should be administered once daily to include a full 21-day cycle. Repeating the 21-day cycle is continued during the treatment with the combination of the invention.

In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered under a non-standard dosing schedule. For example, palbociclib or a pharmaceutically acceptable salt thereof is administered once daily for 14 consecutive days, followed by no treatment for 7 days to include the entire 21-day cycle. Repeating the 21-day cycle is continued during the treatment with the combination of the invention.

The standard clinical dosing regimen for palbociclib or a pharmaceutically acceptable salt thereof is to administer 125 mg once daily for 21 consecutive days, followed by no treatment for 7 days to include a full 28-day cycle. do. Repeating the 28-day cycle is continued during the treatment with the combination of the invention.

In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered under a non-standard dosing regimen. For example, palbociclib or a pharmaceutically acceptable salt thereof is administered once daily at about 50 mg, about 75 mg, or about 100 mg to include a full 28-day cycle. Repeating the 28-day cycle is continued during the treatment with the combination of the invention. In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered at about 50 mg. In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered at about 75 mg. In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered at about 100 mg.

In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered under a non-standard dosing regimen. For example, palbociclib or a pharmaceutically acceptable salt thereof is administered once daily at about 50 mg, about 75 mg, or about 100 mg to include a full 21-day cycle. Repeating the 21-day cycle is continued during the treatment with the combination of the invention. In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered at about 50 mg. In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered at about 75 mg. In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered at about 100 mg.

In one embodiment, palbociclib or a pharmaceutically acceptable salt thereof is administered under a non-standard dosing regimen. For example, palbociclib or a pharmaceutically acceptable salt thereof is administered at a dose of about 75 mg once daily for 14 consecutive days, followed by no treatment for 7 days to include the entire 21-day cycle. Repeating the 21-day cycle is continued during the treatment with the combination of the invention.

In one embodiment of the invention, PF-60952229 may be administered at 20 mg twice daily (BID) and a 7-day treated / 7-day untreated regimen in a 28-day cycle may be employed.

In one embodiment of the invention, PF-60952229 may be administered at 40 mg twice daily (BID) and a 7-day treated / 7-day untreated regimen in a 28-day cycle may be employed.

In one embodiment of the invention, PF-60952229 may be administered at 80 mg twice daily (BID) and a 7-day treated / 7-day untreated regimen in a 28-day cycle may be employed.

In one embodiment of the invention, PF-60952229 may be administered at 150 mg twice daily (BID) and a 7-day treated / 7-day untreated regimen in a 28-day cycle may be employed.

In one embodiment of the invention, PF-60952229 may be administered at 250 mg twice daily (BID) and a 7-day treated / 7-day untreated regimen in a 28-day cycle may be employed.

In one embodiment of the invention, PF-60952229 may be administered at 375 mg twice daily (BID) and a 7-day treated / 7-day untreated regimen in a 28-day cycle may be employed.

In one embodiment of the invention, PF-60952229 may be administered at 500 mg twice daily (BID) and a 7-day treated / 7-day untreated regimen in a 28-day cycle may be employed.

In one embodiment of the invention, PF-60952229 may be administered at 625 mg twice daily (BID) and a 7-day treated / 7-day untreated regimen in a 28-day cycle may be employed.

In a further embodiment of the invention, PF-60952229 is administered in combination with palbociclib and letrozole, where palbociclib is orally administered at 125 mg once daily for 21 days followed by no treatment for 7 days. And letrozole is orally administered at 2.5 mg daily.

Administration of the combination of compounds of the invention can be carried out by any method capable of delivering the compounds to the site of action. These methods include oral, duodenal, parenteral injections (including intravenous, subcutaneous, intramuscular, intravascular, or infusion), topical, and rectal injections.

The compounds of the methods or combinations of the invention can be formulated prior to administration. The formulation is preferably adapted to the particular mode of administration. These compounds can be formulated with pharmaceutically acceptable carriers known in the art and can be administered in a wide range of dosage forms known in the art. In the preparation of the pharmaceutical compositions of the present invention, the active ingredient is usually mixed with, diluted with, or encapsulated in a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, solid diluents or fillers, excipients, sterile aqueous media, and various non-toxic organic solvents. Dosage unit forms or pharmaceutical compositions include capsules such as tablets, gelatin capsules, pills, powders, granules, aqueous and non-aqueous oral solutions and suspensions, medicated drops, troches, hard candy, sprays, creams, ointments, Includes suppositories, jellies, gels, pastes, lotions, ointments, injectable solutions, elixirs, syrups, as well as parenteral solutions packaged in containers suitable for subdivision into individual doses.

Parenteral formulations include pharmaceutically acceptable aqueous or non-aqueous solutions, dispersions, suspensions, emulsions, and sterile powders for their preparation. Examples of carriers include water, ethanol, polyols (propylene glycol, polyethylene glycol), vegetable oils, and organic esters for injection such as ethyl oleate. Fluidity can be maintained by the use of coatings such as lecithin, surfactants, or by maintaining a suitable particle size. Typical parenteral dosage forms include solutions or suspensions of the compounds of the invention in sterile aqueous solutions such as aqueous propylene glycol or dextrose solutions. Such dosage forms can be adequately buffered as needed.

In addition, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are often useful for tablet purposes. Similar types of solid compositions can also be employed in soft and hard filled gelatin capsules. Therefore, preferred materials include lactose or lactose, and high molecular weight polyethylene glycol. If an aqueous suspension or emulsifier is desirable for oral administration, the active compound therein may be a variety of sweets or flavors, colorants or dyes, along with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof. And, if desired, can be combined with emulsifiers or suspending agents.

Methods of preparing various pharmaceutical compositions with a particular amount of active compound will be known or apparent to those of skill in the art. For example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easter, Pa. , 15th Edition (1975).

The invention also relates to a combination of therapeutic agents of the invention, and kits comprising written instructions for administration of the therapeutic agents. In one embodiment, the written instructions detail and define the mode of administration of the therapeutic agent, eg, for simultaneous or continuous administration of the therapeutic agent of the present invention. In one embodiment, the written instructions detail and define the mode of administration of the therapeutic agent, eg, by specifying the number of days of administration for each of the therapeutic agents during a 28-day cycle.

(Example 1) PF-60952229, which is a TGFβ inhibitor, shows synergistic action with the CDK4 / 6 inhibitors palbociclib and CDK2 / 4/6 inhibitor (PF-06673600) in a CT26 syngeneic mouse tumor model. -60952229 was evaluated in combination with palbociclib in a CT26 syngeneic mouse tumor model to evaluate its effectiveness in the growth and survival of primary tumors. PF-069522229 in combination with the CDK4 / 6 inhibitor palbociclib significantly survives compared to PF-069522229 monotherapy (p = 0.009) and palbociclib monotherapy (p = 0.017). Increased.

Materials and Methods CT26 cells were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). All cells were maintained in a humidified incubator at 37 ° C. with 5% carbon dioxide (CO ₂ ). Female Balb / cJ mice were obtained from Jackson Laboratories at 8 weeks of age. To create a syngeneic model, 250,000 CT26 tumor cells were subcutaneously transplanted into the right flank of female BALB / cJ mice. Mice bearing tumors were randomized into 6 treatment groups 10 days after tumor cell transplantation, based on an average tumor size of approximately 50 mm ³ per group. The study group included a medium, a combination of 30 mg / kg PF-60952229, 10 mg / kg PD-0332991 (palbociclib), PF-06673600 (CDK2 / 4/6 inhibitor), PF-60952229 + PD-0332991, and PF-. A combination of 06952229 + PF-06783600 was included. PF-06952229 was orally administered twice daily (BID) on a 7-day treated and 7-day untreated schedule. PD-0332991 or PF-06783600 was orally administered by BID continuously until the end of the study. Information on treatment groups and dosing regimens is summarized in Table 1.

Tumor volume was measured 3 times a week. Tumor volume was calculated based on two-dimensional caliper measurements in cubic millimeter volumes calculated using the formula (length x width ² ) x 0.5. Mice were sacrificed when tumor volume reached 2000 mm ³ , the survival endpoint in this study. Survival curves were plotted using GraphPad Prism 7 software. Statistical analysis was performed using the Logrank (Mantel Cox) test.

result:
The results of survival 40 days after the start of treatment showed that treatment with the TGFβ inhibitor PF-60952229 monotherapy did not significantly increase survival in the CT26 syngeneic tumor model, but with the CDK4 / 6 inhibitor. PF-60952229 treatment in combination with one palbociclib significantly increased survival compared to PF-60952229 monotherapy (p = 0.0088) and palbociclib monotherapy (p = 0.0173). show. Significant combination effects were also seen when the TGFβ inhibitor PF-60952229 was combined with the CDK2 / 4/6 inhibitor PF-06673600, PF-60952229 monotherapy (p <0.0001). And PF-06783600 monotherapy (p = 0.0013) significantly increased survival. See FIGS. 1 and 2.

On day 17 after the start of treatment, the results of tumor growth showed that treatment with the TGFβ inhibitor PF-60952229 monotherapy did not significantly inhibit tumor growth in the CT26 heterologous transplant tumor model. Treatment with PF-06952229 in combination with the CDK2 / 4/6 inhibitor PF-06952229 has a significant combination effect, thus PF-60952229 monotherapy (p = 0.0005) and PF-06673600 monotherapy. It is shown that the inhibition of tumor growth was increased as compared with (p = 0.0004) (Fig. 2). Similarly, the combination of PF-60952229 and palbociclib (PD-0332991) also showed a tendency for combination effects and increased inhibition of tumor growth when compared to treatment alone with PF-60952229 or palbociclib monotherapy. (Fig. 2).

CONCLUSIONS: The combination of the TGFβ inhibitor PF-60952229 with the CDK4 / 6 inhibitor palbociclib or CDK2 / 4/6 inhibitor is PF-069522229 monotherapy or CDK inhibitor monotherapy in the CT26 syngeneic tumor model. Compared with therapy, it increased the inhibition of tumor growth and significantly improved survival.

Example 2 PF-0695222 shows synergistic effects with palbociclib and palbociclib + fulvestrant (PF-05279929) in the MCF7 human ER + heterologous transplant mouse tumor model Overview PF-069522229 MCF-7 ER ^{+ HER2-} In breast cancer tumor mouse model mice, evaluation was performed in the absence or presence of the selective estrogen receptor-degrading agent fulvestrant in combination with the CDK4 / 6 inhibitor palbociclib. The combination of PF-069522229 with the CDK4 / 6 inhibitor palbociclib (PD-0332991) significantly inhibited tumor growth as compared to either monotherapy alone. Similar results were observed when PF-60952229 was combined with palbociclib plus fulvestrant.

Materials and Methods MCF7 human ER ⁺ breast cancer cells were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). All cells were maintained in a humidified incubator at 37 ° C. with 5% carbon dioxide (CO ₂ ). Female NSG mice were obtained from Jackson Laboratories at 7 weeks of age. To create a xenograft model, 17β-ESTRADIOL pellets (0.36 mg, released for 90 days) were subcutaneously transplanted into the left flank of female NSG mice 7 days prior to tumor cell transplantation. Then, 5 million MCF7 cancer cells were subcutaneously transplanted into the right region of the body axis of female NSG mice. Tumor-bearing mice were randomized into treatment groups 27 days after tumor cell transplantation based on an average tumor size of approximately 180 mm ³ and treatment was initiated. The treatment group includes media: PD-0332991 at 10 mg / kg, PF-069522229 at 30 mg / kg, PD-0332991 + PF-05279929 (10 mg / kg), PF-069522229 + PD-0332991, and PF-06952291 + PD-0332991 + PF-05279929. Heavy combinations were included. PF-06952229 was orally administered twice daily (BID) on a 7-day treated and 7-day untreated schedule. PD-0332991 was BID and was orally administered continuously until the end of the study. PF-05279929 was subcutaneously administered twice a week. Information on treatment groups and dosing regimens is summarized in Table 3.

Tumor volume was measured twice a week. Tumor volume was calculated based on two-dimensional caliper measurements in cubic millimeter volumes calculated using the formula (length x width ² ) x 0.5. Body weight was measured twice a week. Tumor growth curves were plotted using GraphPad Prism 7 software. Statistical analysis of the analysis of covariance (ANCOVA) model was applied to assess the effect of treatment on tumor size at each time point after treatment and adjusted for baseline tumor size in individual animals. Comparisons of treatment groups to controls or other treatment groups were performed with t-test under the ANCOVA model with magnification changes to calculate the relevant 95% confidence intervals.

pSMAD2 bioassay: Tumor samples were collected in a 2.0 mL cryotube (Nalgene ™) and snap frozen prior to analysis. Thawed tumor samples were homogenized in cell extraction buffers (Invitrogen, Carlsbad, CA) supplemented with protease and phosphatase inhibitors. Tumor lysates were centrifuged to pellet insoluble debris and the clarified supernatant was transferred to a new tube. PSmad2 was measured using a 6-Plex TGFbeta Signaling Magnetic Bead kit (Millipore, Burlington, MA). All assays were performed at room temperature. After blocking the 96-well black round bottom plate with assay buffer for 10 minutes, 25 μL of working microsphere bead mixture (beads were diluted 1-fold with assay buffer of the kit) and 25 μL of 1:10 diluted tumor lysis. The product (diluted 1:10 with assay buffer) was added to the plate. After incubating overnight with shaking at 4 ° C., the bead mixture was washed using a portable magnetic separation block (EMD Millipore Catalog No. 40-285). The beads bound to pSmad2 were incubated with 25 μL of biotinylated detection antibody solution for 1 hour, then the bead mixture was washed. For detection, 25 μL of streptavidin-PE solution was added and incubated for 15 minutes, then 25 μL of amplification buffer was added and the incubation was repeated for 15 minutes. After washing, the beads were resuspended in a 150 μL / well sheathed solution (Bio-Rad Catalog No. 171-0000055) and analyzed using a Bio-Plex 200 analyzer (Bio-Rad, Hercules CA). The average fluorescence intensity (MFI) of each well was determined using Bio-Plex Manager software, version 6.1 (Bio-Rad). The MFI minus the signal intensity of the blank well was used for further analysis.

Total Smad2 Bioassay: Using the PathScan Total Smad2 Sandwich ELISA Kit (Cell Signaling, Catalog No. 7244C), the total amount of Smad2 protein was determined according to the manufacturer's instructions. Tumor lysate samples were diluted 1: 100 with diluent buffer and 100 μL was added to the appropriate wells. The plates were incubated at 37 ° C. for 2 hours. After washing the plates, the detection solution (100 μL / well) was added and the plates were incubated at 37 ° C. for 1 hour. The plates were washed, then 100 μL of HRP-bound secondary antibody was added and incubated at 37 ° C. for 30 minutes. The plates were washed again, TMB substrate was added and the plates were incubated for 30 minutes at room temperature. To stop the reaction, STOP solution was added to each well. The absorbance of the sample at 450 nm was measured with a Spectramax plate reader (Molecular Devices).

Phospho-Rb Ser807 / 811 Bioassay: Phospho-Rb protein S807 / 811 was analyzed in a multiplex assay in tumor lysates and analyzed into 10-spot 96-well U-PLEX plates purchased from Meso-Scale Discovery (MSD). It was manifested and characterized using a unique linker. The phospho-Rb-specific antibody pS807 / 811 (8516BF) and total Rb antibody (9309BF) were purchased from Cell Signaling Technology (CST). In this 5-PLEX assay, the phospho-Rb-specific antibody was biotinylated and attached to the U-PLEX linker. The linker was then self-assembled as a capture reagent on a unique spot on the U-PLEX plate. Precisely diluted tumor lysates were added to the plate. After the analyte in the sample bound to the capture reagent, the Rb detection antibody conjugated with an electrochemical luminescent label (MSD GOLD SULFO-TAG) bound to the analyte to complete the sandwich immunoassay.

Results On the 21st day after the start of treatment, the results of tumor growth showed that treatment with the TGFβ inhibitor PF-60952229 monotherapy did not significantly inhibit tumor growth in the MCF7 heterologous transplant tumor model. , PF-60952229 treatment in combination with the CDK4 / 6 inhibitor palbociclib has a significant combination effect, thus PF-60952229 monotherapy (p <0.00001) and palbociclib monotherapy (p = 0. It is shown that the inhibition of tumor growth was increased as compared with 0002) (Fig. 3). When combined with palbociclib + fulvestrant, PF-60952229 also showed a significant combined effect of p = 0.0342 compared to palbociclib + fulvestrant treatment (FIG. 4).

On the same day of the study (21 days after treatment initiation), animals in group 2 (palbociclib) were randomized to create two new treatment groups with n = 5 animals per group. Treatment with the TGFβ inhibitor PF-06592229 was then added to one of the newly created groups, and palbociclib treatment was continued until 66 days after the start of treatment for both of the newly created groups. This is the end of the research. The same procedure was performed for group 4 on day 21 and on this day the animals in this group were randomized into two new treatment groups and treatment with the TGFβ inhibitor PF-60952229 was performed on these groups. In addition to one, on the other hand, palbociclib plus fulvestrant treatment continued until day 66 for both newly created groups. Addition of the TGFβ inhibitor PF-069522229 to the palbociclib group or the palbociclib + fulvestrant group had no statistically significant effect compared to palbociclib or palbociclib + fluvestrant alone, but TGFβ inhibition. Treatment with the agent PF-60952229 to the palbociclib or palbociclib plus fluvestrant group tended to cause greater tumor inhibition (FIG. 5).

Biomarker analysis of tumor samples isolated 21 days after the start of treatment showed that treatment with the TGFβ inhibitor PF-06592229 significantly inhibited pSMAD2, a key component of the TGFβ signaling pathway. Was demonstrated (Fig. 6). A modest inhibition of pSMAD2 was also observed in the palbociclib plus fulvestrant group, but the effect of the TGFβ inhibitor PF-60952229 alone was greater than the combination of palbociclib plus fulvestrant (p = 0.004). ) (Fig. 6). The strongest inhibition of pSMAD2 was observed in the group treated with the TGFβ inhibitor PF-60952229 in combination with palbociclib or palbociclib + fulvestrant (about 80% inhibition in both groups), which means that the addition of palbociclib , PSMAD2 levels have been demonstrated to improve the ability of PF-60952229 to downregulate (p = 0.01 and p = 0.007, respectively) (FIG. 6). Phosphorylation of Rb is a downstream biomarker of CDK4 / 6 inhibition in cancer cells. Treatment with palbociclib alone slightly reduced pS807 / 811 Rb levels on day 21, while monotherapy with the TGFβ inhibitor PF-069522229 produced these identical phosphoproteins. It was slightly increased (Fig. 7). An increase in inhibition of pS807 / 811 Rb levels was observed with the combination of palbociclib and fulvestrant (p = 0.04), and a similar improvement was observed with tumors treated with the combination of palbociclib and PF-06952229 (p = 0.04). p = 0.04). Addition of the TGFβ inhibitor PF-60952229 to the palbociclib + fulvestrant combination most strongly inhibited pS807 / 811 Rb levels (p <0.0001) (FIG. 7). Taken together, these data indicate that the use of the TGFβ inhibitor PF-60952229 alone or in combination with palbociclib plus fulvestrant tends to improve inhibition of pS808 / 811 Rb.

CONCLUSIONS: The TGFβ inhibitor PF-069522229 in combination with the CDK4 / 6 inhibitor palbociclib or the palbociclib + selective estrogen receptor-degrading agent fulvestrant is an MCF-7 ER ⁺ HER2 ^- heterologous implant. In a breast cancer tumor model, it resulted in better inhibition of tumor growth compared to PF-60952229 or palbociclib monotherapy or the combination of palbociclib plus fulvestrant. The TGFβ inhibitor PF-60952229 can be added to animals previously treated with the CDK4 / 6 inhibitor palbociclib or palbociclib + fulvestrant for 21 days prior to palbociclib monotherapy or palbociclib + fulvestrant. Compared to the combination, there was a tendency for increased inhibition of tumor growth. In addition, the TGFβ inhibitor PF-60952229 + palbociclib + palbociclib + fulvestrant combination increased inhibition of downstream signaling pathways for both TGFβR1 (pSMAD2) and CDK4 / 6 (pS807 / 811 Rb). rice field.

Claims (8)

A pharmaceutical composition comprising a TGFβ inhibitor for use in the treatment of colon cancer in combination with a CDK inhibitor, wherein the TGFβ inhibitor is 4- (2- (5-chloro-2-fluorophenyl)-. 5-Isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropan-2-yl) nicotine amide or a pharmaceutically acceptable salt thereof, wherein the CDK inhibitor is palbociclib or pharmaceutically acceptable thereof. Salt, or 6- (difluoromethyl) -8-((1R, 2R) -2-hydroxy-2-methylcyclopentyl) -2- (1- (methylsulfonyl) piperidine-4-ylamino) pyrido [2,3- d] A pharmaceutical composition which is pyrimidin-7 (8H) -one or a pharmaceutically acceptable salt thereof. Palbociclib or its pharmaceutically acceptable salt in the treatment of colon cancer,
Approximately 75 mg once daily and / or once daily for 14 consecutive days, followed by no treatment for 7 days.
The pharmaceutical composition according to claim 1. The second aspect of claim 2, wherein in the treatment of colon cancer, about 75 mg of palbociclib or a pharmaceutically acceptable salt thereof is administered once a day for 14 consecutive days, followed by no treatment for 7 days. Pharmaceutical composition. The pharmaceutical composition according to any one of claims 1 to 3, further used in combination with fulvestrant. A pharmaceutical composition comprising a CDK inhibitor for use in the treatment of colon cancer in combination with a TGFβ inhibitor, wherein the CDK inhibitor is palbociclib or a pharmaceutically acceptable salt thereof, or 6- (difluoromethyl). ) -8-((1R, 2R) -2-hydroxy-2-methylcyclopentyl) -2-(1- (methylsulfonyl) piperidine-4-ylamino) pyrido [2,3-d] pyrimidin-7 (8H) -On or a pharmaceutically acceptable salt thereof, the TGFβ inhibitor is 4- (2- (5-chloro-2-fluorophenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3). -Dihydroxypropan-2-yl) A pharmaceutical composition which is a nicotine amide or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 5, which is further used in combination with fulvestrant. A pharmaceutical composition comprising a TGFβ inhibitor for use in the treatment of colon cancer in combination with a CDK4 / 6 inhibitor, wherein the TGFβ inhibitor is 4- (2- (5-chloro-2-fluorophenyl)). -5-Isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropan-2-yl) nicotine amide or a pharmaceutically acceptable salt thereof, and the CDK4 / 6 inhibitor is palbociclib or its pharmaceutically acceptable salt. A pharmaceutical composition that is an acceptable salt. A pharmaceutical composition comprising a TGFβ inhibitor for use in the treatment of colon cancer in combination with a CDK2 / 4/6 inhibitor, wherein the TGFβ inhibitor is 4- (2- (5-chloro-2-fluoro). Phenyl) -5-isopropylpyridine-4-ylamino) -N- (1,3-dihydroxypropane-2-yl) nicotinamide or a pharmaceutically acceptable salt thereof, wherein the CDK2 / 4/6 inhibitor is 6- (Difluoromethyl) -8-((1R, 2R) -2-hydroxy-2-methylcyclopentyl) -2-(1- (methylsulfonyl) piperidine-4-ylamino) pyrido [2,3-d] pyrimidin-7 (8H) -A pharmaceutical composition which is on, or a pharmaceutically acceptable salt thereof.

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